Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. We used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. We have also demonstrated that Brachyury induces an epithelial-to-mesenchymal transition (EMT) in tumor cells, an important step in the progression of primary tumors towards metastasis. Over-expression of Brachyury in human carcinoma cells induces a repertoire of biochemical, morphologic, and functional changes characteristic of EMT. Conversely, inhibition of Brachyury diminishes the ability of human tumor cells to form lung metastases in a xenograft model. An HLA-A0201 epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell mediated cancer immunotherapy. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. We further evaluated for the first time an MVA vector platform for delivery of CD40L to CLL cells (MVA-CD40L) and compared it to MVA-TRICOM for their ability to enhance the immunogenicity of CLL cells in vitro. Our results indicated that MVA-TRICOM-infected and MVA-CD40L-infected CLL cells are equally potent at inducing autologous T-cell proliferation. Therefore, the results from this study further support the rationale for the use of CLL cells modified ex vivo with recombinant MVA as a whole tumor-cell vaccine for the immunotherapy of CLL, either by modification with the MVA-CD40L or MVA-TRICOM vector.